SARS-CoV-2 variants evolve convergent strategies to remodel the host response.

SARS-CoV-2 variants of concern (VOCs) emerged during the COVID-19 pandemic. Here, we used unbiased systems approaches to study the host-selective forces driving VOC evolution. We discovered that VOCs evolved convergent strategies to remodel the host by modulating viral RNA and protein levels, altering viral and host protein phosphorylation, and rewiring virus-host protein-protein interactions. Integrative computational analyses revealed that although Alpha, Beta, Gamma, and Delta ultimately converged to suppress interferon-stimulated genes (ISGs), Omicron BA.1 did not. ISG suppression correlated with the expression of viral innate immune antagonist proteins, including Orf6, N, and Orf9b, which we mapped to specific mutations. Later Omicron subvariants BA.4 and BA.5 more potently suppressed innate immunity than early subvariant BA.1, which correlated with Orf6 levels, although muted in BA.4 by a mutation that disrupts the Orf6-nuclear pore interaction. Our findings suggest that SARS-CoV-2 convergent evolution overcame human adaptive and innate immune barriers, laying the groundwork to tackle future pandemics.

Clinical, immunological and molecular findings of patients with DOCK-8 deficiency from India.

DOCK8 deficiency affects various cell subsets belonging to both the innate and adaptive immune systems. Clinical diagnosis is challenging, as many cases present with severe atopic dermatitis as the only initial manifestation. Though flow cytometry helps in the presumptive diagnosis of DOCK8-deficient patients by evaluating their DOCK8 protein expression, it requires subsequent confirmation by molecular genetic analysis. Currently, haematopoietic stem cell transplantation (HSCT) is the only curative treatment option available for these patients. There is a paucity of data from India on the clinical diversity and molecular spectrum of DOCK8 deficiency. In the present study, we report the clinical, immunological and molecular findings of 17 DOCK8-deficient patients from India diagnosed over the last 5 years.

A Kinematic Analysis of Wrist and Carpal Function Using Four-Dimensional Computed Tomography Technology: A Dynamic Perspective.

PURPOSE: An emerging imaging modality, four-dimensional computed tomography, can provide dynamic evaluation of carpal motion, which allows for a better understanding of how the carpals work together to achieve range of motion. The objective of this work was to examine kinematic motion of the carpus through a flexion/extension arc of motion using four-dimensional computed tomography. METHODS: A convenience sample of 20 uninjured participants underwent a four-dimensional computed tomography scanning protocol through a complete arc of flexion/extension motion. Kinematic changes in motion were quantified using helical axes motion data for each carpal. Rotation angles were compared between bones to identify differences in kinematic motion between bones. RESULTS: The bones within the proximal carpal row, the lunate, scaphoid, and triquetrum, rotate significantly to differing magnitudes at the ends of motion (40° of flexion and 40° of extension). The scaphoid rotates to the highest magnitude, followed by the triquetrum, and lastly, the lunate. The distal carpal row bones rotate to similar magnitudes throughout the entire range of motion. CONCLUSIONS: This work describes the kinematics of the carpals throughout dynamic in vivo flexion and extension. CLINICAL RELEVANCE: This study adds to an understanding of wrist mechanics and the possible clinical implications of pathological deviation from baseline kinematics.

A comprehensive study of p53 protein.

The protein p53 has been extensively investigated since it was found 43 years ago and has become a "guardian of the genome" that regulates the division of cells by preventing the growth of cells and dividing them, that is, inhibits the development of tumors. Initial proof of protein existence by researchers in the mid-1970s was found by altering and regulating the SV40 big T antigen termed the A protein. Researchers demonstrated how viruses play a role in cancer by employing viruses' ability to create T-antigens complex with viral tumors, which was discovered in 1979 following a viral analysis and cancer analog research. Researchers later in the year 1989 explained that in Murine Friend, a virus-caused erythroleukemia, commonly found that p53 was inactivated to suggest that p53 could be a "tumor suppressor gene." The TP53 gene, encoding p53, is one of human cancer's most frequently altered genes. The protein-regulated biological functions of all p53s include cell cycles, apoptosis, senescence, metabolism of the DNA, angiogenesis, cell differentiation, and immunological response. We tried to unfold the history of the p53 protein, which was discovered long back in 1979, that is, 43 years of research on p53, and how p53's function has been developed through time in this article.

Role of Oxidative Stress and the Protective Effect of Fermented Papaya Preparation in Sickle Cell Disease.

Fermented papaya preparation (FPP) is the source of antioxidants that may help in reducing the complications associated with oxidative stress and may improve the quality of life in sickle cell disease patients. In this study, we assessed the in vitro effect of FPP on sickled red blood cells (RBCs) using oxidative stress markers and observed that FPP has the potential to reduce the oxidative stress. Scanning electron microscopy (SEM) and eosin 5' malaemide (E5'M) dye test showed that FPP protects red cell morphology against the oxidative stress. Liquid chromatography mass spectrometry (LCMS) analysis of FPP suggests the presence of essential amino acids, vitamin D3, and its derivatives. Fermented papaya preparation can be of benefit either in reducing oxidative stress parameters or in preventing pathophysiological events in the sickle cell disease patients.

CD8 + T Cells Exhibit an Exhausted Phenotype in Hemophagocytic Lymphohistiocytosis.

PURPOSE: Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory syndrome mainly caused by uncontrolled activation of antigen presenting cells and CD8 T cells. CD8 T cell exhaustion is a known phenomenon in chronic viral infections and cancer. However, the role of T cell exhaustion is not yet identified in HLH in the background of persistent inflammation. So, currently, we have characterized the CD8 T cells using flow cytometry to understand the phenomenon of exhaustion in these cells in HLH. METHODS: We have comprehensively evaluated lymphocyte subsets and characterized CD8 T cells using immunophenotypic markers like PD1, TIM3, LAG3, Ki67, Granzyme B, etc. in a cohort of 21 HLH patients. Effector cytokine secretion and degranulation by CD8 T cells are also studied. RESULTS: Our findings indicate skewed lymphocyte subsets and aberrantly activated CD8 T cells in HLH. CD8 T cells exhibit significantly increased expression of PD1, TIM3, and LAG3 prominently in primary HLH as compared to controls. PD1 + CD8 T cells express elevated levels of Granzyme B and Ki67. Moreover, CD8 T cells are hypofunctional as evidenced by significantly reduced cytokine secretion and compromised CD107a degranulation. CONCLUSION: The study has revealed that CD8 + cytotoxic T lymphocytes from HLH patients exhibited high expression of exhaustion markers with overall impaired function. To the best of our understanding, this is the first report suggesting functional exhaustion of CD8 T cells in both primary and secondary HLH. Future studies to understand the association of exhaustion with disease outcome are needed for its probable therapeutic implementation.

Fabrication of gelatin functionalized silver nanoparticles for blood group profiling.

We report the fabrication of silver nanoparticles (AgNPs) surface functionalized with gelatin at different concentrations (G10/G20/G40 AgNPs) with an average particle size of ∼200 nm, bioconjugated with antisera antibodies (AsAbs) of the major and clinically significant blood groups (CSBGs) at different titres from neat to 1:128. Bioconjugation using ionic interaction at pH 7.4 enabled 'end-on' configuration, with the -NH2 group of the antibody free for interaction with the red blood cell antigen, as confirmed by Fourier transform infrared spectroscopy. The tube agglutination test (TAT) revealed optimum agglutination with G20NPs, while SDS PAGE confirmed the optimal titre as 1:8 for the major blood groups A, B, AB and O. Bioconjugated AgNPs coated onto microtitre assay plates with the major blood groups and CSBGs to enable simultaneous identification, were validated against the TAT on 400 random blood samples for the major blood groups and revealed high accuracy (95%). While similar accuracy was seen for most of the CSBGs with only false negatives, the method was not found to be suitable for the Kell, Kidd and Duffy groups. The absence of false positives reflects high safety, and eliminates the risk of a mismatched blood transfusion. The method uses diluted blood and hence could enable point-of-care detection. The significantly lower AsAb requirement also provides a cost advantage.

Influence of Sex and Previous Concussion History on Postconcussive Recovery in Young Athletes.

OBJECTIVE: To determine whether concussed female athletes with a previous history of concussion have longer postconcussive recovery than that of male counterparts. DESIGN: Retrospective cohort study. SETTING: Outpatient sports medicine clinic in an academic practice. PARTICIPANTS: Male and female youth, high school, and collegiate athletes (n = 431; ages = 10-21 years) who sustained a sport-related concussion (SRC). INTERVENTIONS: The clinical courses of young athletes treated for concussion by 1 provider at an outpatient sports medicine clinic were retrospectively reviewed. MAIN OUTCOME MEASURES: Recovery time was compared after an SRC with relationship to sex and previous concussion history. RESULTS: When comparing male and female athletes with a previous history of concussion, there were no differences found (P = 0.820) in SRC recovery time. Regardless of previous concussion history, males recovered faster from an SRC compared with their female counterparts (P = 0.0002). Without regard to sex, those with no previous history of concussion recovered faster than those with a previous concussion history, although the difference was not statistically significant (P = 0.668). Athletes with a previous history of concussion were more likely to require neuropsychology referral than those with no previous concussion history (P = 0.021), and females, without regard to concussion history, were more likely to require neuropsychology referral than males (P = 0.001). CONCLUSIONS: A previous concussion history does not appear to significantly influence postconcussive recovery time in young athletes, although it does increase the probability of neuropsychological referral. Without regard to a previous concussion history, young female athletes recover slower than males from concussion and are also more likely to require neuropsychological referral.

Organocatalyzed Domino Synthesis of New Thiazole-Based Decahydroacridine-1,8-diones and Dihydropyrido[2,3-d : 6,5-d']- dipyrimidines in Water as Antimicrobial Agents.

Organopromoter, 2-aminoethanesulfonic acid was used to catalyze the synthesis of a series of structurally intriguing new hybrids thiazolyl acridine-1,8(2H,5H)-diones and dihydropyrido[2,3-d : 6,5-d']dipyrimidine-2,4,6,8(1H,3H,5H,7H)-tetraones for the first time. 2-Aminoethanesulfonic acid is a biobased organopromoter, used to generate four new bonds for the synthesis of new coupled thiazole-based decahydroacridine-1,8-diones. Superior green credentials, operational simplicity, easy work-up and recyclability of the catalyst are the key strengths of this method. The broad substrate scope, mild reaction conditions, short reaction time, cost effectiveness, high atom economy and good to excellent yields make the present method a distinct improvement over existing methods. Spectral (IR, 1 H-NMR,13 C-NMR, Mass) data and elemental analyses confirmed the structures of the titled products. A series of thiazolyl acridine-1,8(2H,5H)-diones and dihydropyrido[2,3-d : 6,5-d']dipyrimidine-2,4,6,8(1H,3H,5H,7H)-tetraones were screened for their antimicrobial activity against four bacterial and three fungal strains.

Fibrotic Cytokine Interplay in Evaluation of Disease Activity in Treatment Naïve Systemic Sclerosis Patients from Western India.

BACKGROUND: : Systemic sclerosis (SSc) is a demyelinating disease of skin, subcutaneous tissue, muscles and internal organs, with fibrosis as an important pathological event. AIM: : To understand cytokine interplay of IL-1ß, IL-4 and IL-6 and their association with disease activity in treatment naïve active cases of systemic sclerosis from Western India. METHODS: Twenty-five SSc patients as per ACR-EULAR 2013 criteria (classified based on pulmonary fibrosis and generalized fibrosis) and 25 age-sex matched controls were enrolled. Serum cytokine levels of IL-1ß, IL-4 and IL-6 were assessed by multiplex bead based immunoassay. RESULTS: Ten patients had Interstitial lung disease (ILD), whereas, 16 patients had generalized fibrosis. Anti-nuclear antibodies were seen in 22 patients (88%); antiScl70 in 15 patients (60%) and anti-Centromere antibodies in 5 patients (20%). Serum levels of IL-1ß in patients were significantly higher than healthy controls (p=0.0006). IL-4 levels in all SSc patients were marginally raised (p=0.0102), while IL-6 levels were significantly raised (p<0.0001). IL-4 was found to be significantly raised in SSc patients with ILD (p=0.021) as compared to patients without ILD. IL-1ß (p=0.0293) and IL-4 (p<0.0001) were significantly higher in SSc patients with fibrosis. On the contrary, IL-6 levels in patients with fibrosis were found to be lower than in patients without fibrosis. CONCLUSION: Significantly raised cytokine levels among treatment naïve systemic sclerosis patients were found to be associated with higher disease severity in our study. Higher levels of IL-1ß and IL-6 indicated an active inflammatory status, whereas significantly raised IL-4 levels indicated at higher fibrotic activity.

Castleman Disease Presenting with Pseudotumour Cerebri and Myasthenia Gravis: A Case Report and Literature Review.

Castleman disease (CD) is a rare lymphoproliferative disorder that may present with various autoimmune, inflammatory, or neurologic syndromes. This is a case of a 21-year-old woman who presented with signs and symptoms of pseudotumour cerebri (PTC) who subsequently developed myasthenia gravis (MG), and was incidentally found to have a large mass in the posterior mediastinum. Upon resection, the mass was classified as unicentric CD involved with follicular dendritic cell sarcoma. Following treatment with IVIG in the setting of progressive weakness and dyspnea, she has had complete symptom resolution while maintained on a low dose of pyridostigmine for the last two years. There are 13 cases of MG and five cases of optic disc edema described as PTC associated with CD in the literature, but to our knowledge, this is the sole case reported of the intersection of all three conditions in one patient. Increased serum levels of interleukin-6 and vascular endothelial growth factor may provide clues as to the association of CD with these neurologic syndromes.

RsmV, a Small Noncoding Regulatory RNA in Pseudomonas aeruginosa That Sequesters RsmA and RsmF from Target mRNAs.

The Gram-negative opportunistic pathogen Pseudomonas aeruginosa has distinct genetic programs that favor either acute or chronic virulence gene expression. Acute virulence is associated with twitching and swimming motility, expression of a type III secretion system (T3SS), and the absence of alginate, Psl, or Pel polysaccharide production. Traits associated with chronic infection include growth as a biofilm, reduced motility, and expression of a type VI secretion system (T6SS). The Rsm posttranscriptional regulatory system plays important roles in the inverse control of phenotypes associated with acute and chronic virulence. RsmA and RsmF are RNA-binding proteins that interact with target mRNAs to control gene expression at the posttranscriptional level. Previous work found that RsmA activity is controlled by at least three small, noncoding regulatory RNAs (RsmW, RsmY, and RsmZ). In this study, we took an in silico approach to identify additional small RNAs (sRNAs) that might function in the sequestration of RsmA and/or RsmF (RsmA/RsmF) and identified RsmV, a 192-nucleotide (nt) transcript with four predicted RsmA/RsmF consensus binding sites. RsmV is capable of sequestering RsmA and RsmF in vivo to activate translation of tssA1, a component of the T6SS, and to inhibit T3SS gene expression. Each of the predicted RsmA/RsmF consensus binding sites contributes to RsmV activity. Electrophoretic mobility shifts assays show that RsmF binds RsmV with >10-fold higher affinity than RsmY and RsmZ. Gene expression studies revealed that the temporal expression pattern of RsmV differs from those of RsmW, RsmY, and RsmZ. These findings suggest that each sRNA may play a distinct role in controlling RsmA and RsmF activity.IMPORTANCE The members of the CsrA/RsmA family of RNA-binding proteins play important roles in posttranscriptional control of gene expression. The activity of CsrA/RsmA proteins is controlled by small noncoding RNAs that function as decoys to sequester CsrA/RsmA from target mRNAs. Pseudomonas aeruginosa has two CsrA family proteins (RsmA and RsmF) and at least four sequestering sRNAs (RsmV [identified in this study], RsmW, RsmY, and RsmZ) that control RsmA/RsmF activity. RsmY and RsmZ are the primary sRNAs that sequester RsmA/RsmF, and RsmV and RsmW appear to play smaller roles. Differences in the temporal and absolute expression levels of the sRNAs and in their binding affinities for RsmA/RsmF may provide a mechanism of fine-tuning the output of the Rsm system in response to environmental cues.

Functional Analyses of the RsmY and RsmZ Small Noncoding Regulatory RNAs in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen with distinct acute and chronic virulence phenotypes. Whereas acute virulence is typically associated with expression of a type III secretion system (T3SS), chronic virulence is characterized by biofilm formation. Many of the phenotypes associated with acute and chronic virulence are inversely regulated by RsmA and RsmF. RsmA and RsmF are both members of the CsrA family of RNA-binding proteins and regulate protein synthesis at the posttranscriptional level. RsmA activity is controlled by two small noncoding regulatory RNAs (RsmY and RsmZ). Bioinformatic analyses suggest that RsmY and RsmZ each have 3 or 4 putative RsmA binding sites. Each predicted binding site contains a GGA sequence presented in the loop portion of a stem-loop structure. RsmY and RsmZ regulate RsmA, and possibly RsmF, by sequestering these proteins from target mRNAs. In this study, we used selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) chemistry to determine the secondary structures of RsmY and RsmZ and functional assays to characterize the contribution of each GGA site to RsmY/RsmZ activity. Our data indicate that RsmA has two preferential binding sites on RsmY and RsmZ, while RsmF has one preferential binding site on RsmY and two sites on RsmZ. Despite RsmF and RsmA sharing a common consensus site, RsmF binding properties are more restrictive than those of RsmA.IMPORTANCE CsrA homologs are present in many bacteria. The opportunistic pathogen Pseudomonas aeruginosa uses RsmA and RsmF to inversely regulate factors associated with acute and chronic virulence phenotypes. RsmA has an affinity for RsmY and RsmZ higher than that of RsmF. The goal of this study was to understand the differential binding properties of RsmA and RsmF by using the RsmY and RsmZ regulatory small RNAs (sRNAs) as a model. Mutagenesis of the predicted RsmA/RsmF binding sites on RsmY and RsmZ revealed similarities in the sites required to control RsmA and RsmF activity in vivo Whereas binding by RsmA was relatively tolerant of binding site mutations, RsmF was sensitive to disruption to all but two of the sites, further demonstrating that the requirements for RsmF binding activity in vivo and in vitro are more stringent than those for RsmA.

What Is the Optimal Criteria to Use for Detecting Periprosthetic Joint Infections Before Total Joint Arthroplasty?

BACKGROUND: The purposes of this study were to (1) test the accuracy of α-defensin and combined α-defensin-aspiration cultures in diagnosing periprosthetic joint infection (PJI) before revision total knee and hip arthroplasty and (2) evaluate Musculoskeletal Infection Society (MSIS) criteria and α-defensin as predictors of successful reimplantation (second-stage) at 1 year after surgery. METHODS: We retrospectively evaluated a total of 97 synovial fluid aspirations performed between August 2014 and September 2016 before revision due to septic or aseptic failures (n = 70) or before second-stage (n = 27) joint arthroplasty. Revisions were categorized as either septic or aseptic according to the MSIS criteria. Synovial fluid was tested for α-defensin, cell count with differential, and cultures. Reimplantations were assessed for success or failure (defined as the need for reoperation due to infection) within 1 year after surgery. RESULTS: For septic and aseptic revision arthroplasty, the sensitivity, specificity, positive predictive value, and negative predicted value of α-defensin was 97% while for the combined α-defensin and aspiration culture, it was 96%, 100%, 100%, and 97%. Despite being performed with negative MSIS criteria and α-defensin test results, 11% (3/27) of reimplantations (second-stage) failed within 1 year postoperatively because of infection. CONCLUSION: Alpha-defensin is an accurate diagnostic test for the diagnosis of PJI before revision arthroplasty. The combination of α-defensin and aspiration cultures has higher specificity and positive predictive value. MSIS criteria and α-defensin may help predict the success of reimplantations within 1 year after surgery.

Using a Model to Understand the Symptoms of Ophthalmoplegia.

When the term muscular paralysis is used, most people will think of large muscle groups such as the upper and lower limbs or life-dependent muscles such as the diaphragm. However, the extrinsic extraocular muscles can also succumb to paralysis (whether partial or otherwise). Ophthalmoplegia can arise from a number of neural conditions, and in conjunction with the complex anatomy of the ocular orbit, it can be difficult to teach such syndromes. The range of existing physical models for the eye are limited in their functional ability, prohibiting the understanding of the structure and its function especially with regards to muscles. Only one eye model has been developed which is tangible and functional by design in relation to rotational movements (Williams, 1965). The aim of this study was to ultimately build a modern version of Williams' model and via means of a Likert-type, cross-sectional questionnaire, determine the model's capacity to assist students in learning the function and anatomy of the extrinsic muscles of the eye. This foundational knowledge could then be transferred to better understand the internal causes of the visible symptoms of ophthalmoparesis and ophthalmoplegia. In much the same way that different diagnostic scans are used to observe different bodily materials, functional models may not necessarily replace the range of anatomical resources which exist, but it is hoped that models such as this will instead provide insight into an alternative aspect of anatomical learning which is yet to be considered.

Primary and Secondary Sequence Structure Requirements for Recognition and Discrimination of Target RNAs by Pseudomonas aeruginosa RsmA and RsmF.

UNLABELLED: CsrA family RNA-binding proteins are widely distributed in bacteria and regulate gene expression at the posttranscriptional level. Pseudomonas aeruginosa has a canonical member of the CsrA family (RsmA) and a novel, structurally distinct variant (RsmF). To better understand RsmF binding properties, we performed parallel systematic evolution of ligands by exponential enrichment (SELEX) experiments for RsmA and RsmF. The initial target library consisted of 62-nucleotide (nt) RNA transcripts with central cores randomized at 15 sequential positions. Most targets selected by RsmA and RsmF were the expected size and shared a common consensus sequence (CANGGAYG) that was positioned in a hexaloop region of the stem-loop structure. RsmA and RsmF also selected for longer targets (≥96 nt) that were likely generated by rare PCR errors. Most of the long targets contained two consensus-binding sites. Representative short (single consensus site) and long (two consensus sites) targets were tested for RsmA and RsmF binding. Whereas RsmA bound the short targets with high affinity, RsmF was unable to bind the same targets. RsmA and RsmF both bound the long targets. Mutation of either consensus GGA site in the long targets reduced or eliminated RsmF binding, suggesting a requirement for two tandem binding sites. Conversely, RsmA bound long targets containing only a single GGA site with unaltered affinity. The RsmF requirement for two binding sites was confirmed with tssA1, an in vivo regulatory target of RsmA and RsmF. Our findings suggest that RsmF binding requires two GGA-containing sites, while RsmA binding requirements are less stringent. IMPORTANCE: The CsrA family of RNA-binding proteins is widely conserved in bacteria and plays important roles in the posttranscriptional regulation of protein synthesis. P. aeruginosa has two CsrA proteins, RsmA and RsmF. Although RsmA and RsmF share a few RNA targets, RsmF is unable to bind to other targets recognized by RsmA. The goal of the present study was to better understand the basis for differential binding by RsmF. Our data indicate that RsmF binding requires target RNAs with two consensus-binding sites, while RsmA recognizes targets with just a single binding site. This information should prove useful to future efforts to define the RsmF regulon and its contribution to P. aeruginosa physiology and virulence.

Synthesis and antitubercular activity of new 1,3,4-oxadiazoles bearing pyridyl and thiazolyl scaffolds.

In search of more potent and safe new antitubercular agents, here new 2-pyridinyl substituted thiazolyl-5-aryl-1,3,4-oxadiazoles (6a-o), have been designed and synthesized using thionicotinamide as a starting, following novel multistep synthetic route. An intermediate, pyridinyl substituted thiazolyl acid hydrazide (4) when condensed with benzoic acids/nicotinic acids (5a-o) in the presence of silica supported POCl3 yielded better to excellent yields of the title compounds. All the synthesized compounds (6a-o) and intermediate acid hydrazide (4) have been screened for their in vitro antitubercular activity against Mycobacterium tuberculosis H37Ra (MTB) and Mycobacterium bovis BCG. Amongst them, 6f, 6j, 6l and 6o have revealed promising activity against M. bovis BCG at concentrations less than 3µg/mL. These compounds have shown low cytotoxicity (CC50: >100µg/mL) towards four human cancer cell lines. Molecular docking study has also been performed against mycobacterial enoyl reductase (InhA) enzyme to gain an insight into the binding modes of these molecules and recorded good binding affinity. The ADME properties the title products have also been analyzed.

An unusual CsrA family member operates in series with RsmA to amplify posttranscriptional responses in Pseudomonas aeruginosa.

Members of the CsrA family of prokaryotic mRNA-binding proteins alter the translation and/or stability of transcripts needed for numerous global physiological processes. The previously described CsrA family member in Pseudomonas aeruginosa (RsmA) plays a central role in determining infection modality by reciprocally regulating processes associated with acute (type III secretion and motility) and chronic (type VI secretion and biofilm formation) infection. Here we describe a second, structurally distinct RsmA homolog in P. aeruginosa (RsmF) that has an overlapping yet unique regulatory role. RsmF deviates from the canonical 5 ß-strand and carboxyl-terminal α-helix topology of all other CsrA proteins by having the α-helix internally positioned. Despite striking changes in topology, RsmF adopts a tertiary structure similar to other CsrA family members and binds a subset of RsmA mRNA targets, suggesting that RsmF activity is mediated through a conserved mechanism of RNA recognition. Whereas deletion of rsmF alone had little effect on RsmA-regulated processes, strains lacking both rsmA and rsmF exhibited enhanced RsmA phenotypes for markers of both type III and type VI secretion systems. In addition, simultaneous deletion of rsmA and rsmF resulted in superior biofilm formation relative to the wild-type or rsmA strains. We show that RsmF translation is derepressed in an rsmA mutant and demonstrate that RsmA specifically binds to rsmF mRNA in vitro, creating a global hierarchical regulatory cascade that operates at the posttranscriptional level.

Synthesis of new thiazolylmethoxyphenyl pyrimidines and antihyperglycemic evaluation of the pyrimidines, analogues isoxazolines and pyrazolines.

New thiazolylmethoxyphenyl pyrimidines (7a-g) have been conveniently synthesized with better yields by cyclocondensing 3-(4-((2-phenylthiazol-4-yl)methoxy)phenyl)-1-(4-substituted phenyl)prop-2-en-1-ones (4a-g) with thiourea in aqueous emulsion of tetradecyltrimethylammonium bromide (TTAB) at 80 °C. Antihyperglycemic activity of the new thiazolylmethoxyphenyl pyrimidines (7a-d), thiazolylmethoxyphenyl pyrazolines (5a-d) and thiazolylmethoxyphenyl isoxazolines (6a-d) has been evaluated in sucrose loaded rat model. Among these compounds; 5a, 5c, 6b, 7c and 7d have displayed noticeable antihyperglycemic activity. Pyrimidines and pyrazolines have displayed better antihyperglycemic activity than the analogues isoxazolines.

The AlgZR two-component system recalibrates the RsmAYZ posttranscriptional regulatory system to inhibit expression of the Pseudomonas aeruginosa type III secretion system.

Pseudomonas aeruginosa causes chronic airway infections in cystic fibrosis (CF) patients. A classic feature of CF airway isolates is the mucoid phenotype. Mucoidy arises through mutation of the mucA anti-sigma factor and subsequent activation of the AlgU regulon. Inactivation of mucA also results in reduced expression of the Vfr transcription factor. Vfr regulates several important virulence factors, including a type III secretion system (T3SS). In the present study, we report that ExsA expression, the master regulator of T3SS gene expression, is further reduced in mucA mutants through a Vfr-independent mechanism involving the RsmAYZ regulatory system. RsmA is an RNA binding protein required for T3SS gene expression. Genetic experiments suggest that the AlgZR two-component system, part of the AlgU regulon, inhibits ExsA expression by increasing the expression of RsmY and RsmZ, two small noncoding RNAs that sequester RsmA from target mRNAs. Epistasis analyses revealed that increasing the concentration of free RsmA, through either rsmYZ deletion or increased RsmA expression, partially restored T3SS gene expression in the mucA mutant. Furthermore, increasing RsmA availability in combination with Vfr complementation fully restored T3SS expression. Recalibration of the RsmAYZ system by AlgZR, however, did not alter the expression of other selected RsmA-dependent targets. We account for this observation by showing that ExsA expression is more sensitive to changes in free RsmA than other members of the RsmA regulon. Together, these data indicate that recalibration of the RsmAYZ system partially accounts for reduced T3SS gene expression in mucA mutants.